Over the last several years, many humanized or fully human antibodies have been tested in clinical trials, and several of them have been approved for therapeutic application (Hoogenboom, H. R. (2005), Nat Biotechnol 23, 1105-1116; Carter, P. J. (2006). Nat Rev Immunol 6, 343-357). Although the human antibodies typically selected by phage display (Hoogenboom, 2005) are not likely to be immunogenic and therefore quickly neutralized in human, they usually suffer from rapid clearance in experimental animals due to induction of anti-human antibodies. The rapid clearance of human antibodies prevents long-term target-validation, mechanistic and ADME (PK/PD) preclinical studies in rodents. For these reasons, it is highly desirable to have available antibodies which are not immunogenic in the respective animal models. Most preferably, such antibodies are fully derived from the respective animal species. For example, an antibody used in murine animal experiments is ideally a fully murine antibody. Likewise, an antibody used in rat animal experiments is ideally a fully rat antibody.
For the generation of human antibodies synthetic combinatorial antibody libraries have been used, e.g. the HuCAL GOLD library described in Rothe et al., 2008, J Mol Biol 376, 1182-1200. The selection by phage display of well expressed, target-selective high affinity-binders was demonstrated in these fully human library (Steidl et al. 2008. Mol Immunol 46(1):135-44). Synthetic combinatorial libraries offer several advantages over libraries derived from naïve B-cell sources. Natural occurring gene sequences encoding antibody frameworks can be chosen and readily designed for high antibody-production yield in prokaryotic or eukaryotic hosts, e.g. by codon-optimization. Also, the synthetic nature of the libraries enables the implementation of other features, e.g. a modular design of the antibody frameworks with unique restriction sites. Such willfully introduced restriction sites at appropriate positions enable downstream optimization processes such as antibody maturation; for example, pools of binders can be rapidly optimized without knowledge of particular sequences using pre-built diversified complementarity determining region (CDR) cassettes. Mostly this optimization process is driven for a higher antigen affinity and then known as affinity maturation (Steidl et al. 2008. Mol Immunol 46(1):135-44).
A similar approach was not deemed to be possible for rodent antibody libraries. Numerous reports demonstrate the belief in the scientific community that, e.g., mouse antibody libraries cannot be produced with sufficient complexity and/or with reasonably useful expression levels. Historically, mouse antibody-libraries were derived by PCR amplification of a VH-gene pool from spleens of immunized mice (Ward et al., 1989, Nature 341, 544-546). However, such libraries were fraught with problems, such as for example poor expression of the library members.
Only recently, the first synthetic mouse antibody library has been reported (Cobaugh et al., 2008, Journal of Molecular Biology 378, 622-633). But there, the library was focused towards peptide binders, based on single framework derived from antibody 26-10, and highly diversified in HCDR3 only.
WO 06/030238 describes the “surprising discovery that an antibody framework region based on a murine VH14 heavy chain and a murine VK2 light chain [ . . . ] is solubly expressed in a microorganism and is stable [ . . . ]”. See lines 15-28 on page 3 of WO 06/030238. This summarizes all previous, failed attempts in the art to come up with similar murine or other rodent libraries. Notably, WO 06/030238 only reported on the soluble expression of one—and only one—particular VH/VL pair. Accordingly, no other rodent libraries were reported so far as having been successful. Further, no rodent antibody library comprising more than one specific VH/VL pair has been disclosed.
Despite these discouraging references, the present inventors aimed for the generation of antibody libraries of a rodent nature, such as a fully murine and fully rat nature, respectively, and/or collectively. Such libraries could prove suitable for the selection of fully murine and rat antibodies with desired biophysical properties and target specificity. One of the main aims was to build a synthetic fully murine combinatorial antibody library (hereinafter HuCAL MOUSE) and a fully rat combinatorial antibody library (hereinafter HuCAL RAT), preferably comprising as many distinct VL/VH-frameworks as possible.
WO 01/92291 describes a method for the RNA trans-splicing mediated covalent intracellular fusion of transcripts of two different genes. In Example 9a mouse library of dTS (double trans splicing) genes is constructed by comparing and the leader exons of 41 functional mouse VH genes. No mouse antibody libraries were however generated.
De Jaeger et al (FEBS Letters (1997) 403, 116-22) use a single-chain murine phage display library for the isolation of binders against an enzyme from Petunia hybrida. This library is however not a synthetic library. Furthermore the library of de Jaeger et al. is derived from immunized mice, i.e. the library was generated from nucleic acid material that has been pre-exposed to the antigen.
Sommavilla et al (J Immunol Methods (2010) 353, 31-43) report the design and construction of a naive mouse antibody library. Like other attempts, the libraries of Sommavilla et al. only contain one VH and one VL germline gene.
To the inventors' knowledge, neither a synthetic rodent antibody library nor any other reliable rodent antibody library with a diverse VH/VL composition previously has been disclosed which comprises members of more than one VH and/or more than one VL germline family. One of the breakthroughs in the instant invention is based on the observation that different frameworks impose distinct conformations on CDRs, and hence affect the range of antibody-structures capable of antigen binding. This is one of the main problems in the generation of rodent antibodies, and one of the reasons why up until now no rodent antibody library as contemplated by the present invention has been generated. For the first time it was possible overcome the prejudice in the prior art. Rational analysis of the naturally occurring rodent antibody sequences and sophisticated design of the libraries, led to the first synthetic rodent antibody libraries that are broadly useful for biomedical research.